Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Clarified cell-free extracts were prepared from rapidly dividing $\text{Bacillus subtilis}$ cells and from rabbit liver cells. These extracts were treated with [³H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the $\text{B. subtilis}$ extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.     

Direct binding studies do not support the existence of true alpha-adreno-receptors in rat white fat cells

The complexity of mitochondrial translation products in mouse liver and Ehrlich ascites tumor cells have been studied using a mitoplast system active in ³⁵S methionine incorporation. Electrophoretic analysis on gradient polyacrylamide-SDS gels and urea-SDS gels under highly dissociating conditions show that both of the mitochondrial systems synthesize about 22 polypeptides. Many of these ³⁵S labeled products compare with the polypeptides predicted by the DNA sequence analysis data reported by Anderson $\text{et al.}$ (1).     

Identification of the regulatory subunit of a cAMP-dependent protein kinase in Dictyostelium discoideum

The occurrence of a cytosolic cAMP-binding protein of an approximate molecular weight of 41,000 daltons was monitored in vegetative and developing amoebae of $\text{Dictyostelium}\text{discoideum}$ by the use of the photoaffinity probe (³²P) 8N₃-cAMP. There was a large apparent increase in the amount of this binding protein during development; its molecular weight remained constant, if appropriate methods were employed for the disruption of the amoebae. Comigration during electrophoresis on two-dimensional gels identifies this cAMP-binding protein, photoaffinity-labeled in crude extracts, as the regulatory subunit of the cAMP-dependent protein kinase of $\text{D.}\text{discoideum}$.     

Prolonged lifetime of the 410-nm intermediate of bacteriorhodopsin in the presence of guanidine hydrochloride.

Prostatic binding protein (PBP) is a quantitatively important steroid-binding protein present in rat ventral prostate. Electrophoresis on SDS-containing polyacrylamide gels shows that PBP is composed of two subunits, F and S having molecular weights of 16,000 and 18,000. Upon reduction these subunits dissociate further into smaller components. Translation of mRNA from rat ventral prostate in a wheat germ cell-free system or in $\text{Xenopus}$ oocytes results in the formation of polypeptides immunoprecipitable with an anti-PBP antiserum. However, as opposed to the wheat germ system, only the oocytes synthesize polypeptides, that are electrophoretically identical to those of native cytosolic PBP.     

Tubulin-like protein from Aspergillus nidulans

[³⁵S] labeled extracts of the fungus $\text{Aspergillus nidulans}$ were copolymerized with purified porcine brain tubulin. The [³⁵S] $\text{A. nidulans}$ protein which copurified with porcine microtubules was found to be similar to [³H] chick tubulin when the two were coelectrophoresed on several polyacrylamide gel electrophoresis systems. These results strongly suggest the presence in $\text{A. nidulans}$ of a tubulin-like protein.     

Thylakoid membrane protein phosphorylation in correlation with photosynthetic membrane activation

The phosphorylation of five $\text{E.}\text{gracilis}$ thylakoid membrane polypeptides was studied, in isolated chloroplasts. Using [³²P] labelling, in the light, we found that phosphorylation was inhibited by ethanol and DCMU. Inhibition curves were characteristic of photosynthetic inhibition. [γ-³²P] ATP labelling was used to distinguish between two groups of phosphoproteins: the first one, includes protein I, II, V which require only ATP for phosphorylation while the second one includes protein III and IV whose phosphorylation is light-requiring. Phosphorylation of protein III and IV was inhibited by CCCP, NH₄Cl and DCMU, and was reversible in the dark.     

The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme

A strong proteolytic activity is unmasked and solubilized when $\text{E. coli}$ outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of $\text{E.coli}$ located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme.     

Turnover of prolyl hydroxylase and an immunologically related protein in rabbit tissue

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [³H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH₄)SO₄ supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for prolyl hydroxylase of 73 h and a $\text{1}\text{2}$ for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true $\text{t}\text{1}\text{2}$ for prolyl hydroxylase of 45 h was determined. The $\text{t}\text{1}\text{2}$ values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κ_d values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein.     

Radioimmunoassay for γ-endorphin

A double antibody radioimmunoassay technique for γ-endorphin has been developed. The antisera have been raised in rabbits against synthetic γ-endorphin coupled to bovine serum albumin by carbodiimide. The best antibody has a working titer of $\text{1}\text{35,000}$ and can detect less than 9 pg of peptide. The usable range of the standard curve is between 9 to 2400 pg. This antiserum probably binds the Glu⁸-Leu¹⁷ region of γ-endorphin and shows only weak cross-reactivity with α-endorphin, β-endorphin and β-lipotropin. Parallelism is observed between the standard curve and the inhibition curves obtained with rat neurohypophysis-pars intermedia extracts or rat plasma.     

Changes in order of migration of polypeptides in complex III and cytochrome C oxidase under different conditions of SDS polyacrylamide gel electrophoresis.

The order of migration of polypeptides in both cytochrome $\text{c}$ oxidase and ubiquinone cytochrome $\text{c}$ reductase has been found to differ depending on the gel conditions used. Thus the nomenclatures or numbering systems being used for the subunits of these membrane complexes by workers using Weber-Osborn gels is not the same as that being used in laboratories which use the Swank-Munkres or Fairbanks gel procedure.     

Adaptive synthesis of rat liver fatty acid synthetase: evidence for in vitro formation of active enzyme from inactive protein precursors and 4'-phosphopantetheine

Evidence is presented in support of the hypothesis that an important step in the adaptive synthesis of fatty acid synthetase is the conversion of inactive enzyme precursors to active enzyme via the incorporation of the 4′-phosphopantetheine prosthetic group. Fatty acid synthetase activity was generated $\text{in vitro}$ when CoA or $\text{E. coli}$ acyl carrier protein was incubated with enzymatically inactive extracts from livers of rats fed a fat-free diet for 0–5 hr following starvation, and a factor present in liver extracts from rats refed for more than 6 hr. When (¹⁴C)-CoA, labelled in the pantetheine moiety, was used in the above system, radioactivity was incorporated into a protein bound form, from which it could be released by mild alkaline hydrolysis.     

Absence of ferric enterobactin receptor modification activity in mutants of Escherichia coli K-12 lacking protein a.

The modification activity for the ferric enterobactin receptor in the Triton X-100 solubilized outer membrane of $\text{Escherichia}\text{coli}$ K-12 was adsorbed to a column of $\text{p}$-aminobenzamidine-//-sepharose and eluted with free benzamidine. Recombination of the dialyzed eluate with the filtrate from the column reinstituted conversion of the receptor from 81K to 81K¹, the latter exhibiting an apparent molecular weight of 74,000 daltons in sodium dodecyl sulfate polyacrylamide gel analysis. The eluate from the $\text{p}$-aminobenzamidine column was shown to contain a component, coincident on gels with both protein and modification activity, which by mutational and other analyses appears to be identical with protein $\text{a}$ of the outer membrane.     

Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Purification of the prothoracicotropic hormone from the tobacco hornworm Manducasexta

Standard biochemical procedures were used to purify the prothoracicotropic hormone (PTTH) 4400 fold from whole head extracts of $\text{Mandura}$$\text{sexta}$ fifth instar larvae. Hormonal activity was bioassayed by injection into neck-ligated fourth instar larvae. The hormone was stable to heating at 85°C. Ammonium sulfate and acetone fractionation provided a crude preparation which showed dose-dependent activity in the bioassay. Chromatography on Sephadex G-100, DEAE-Sephadex, and hydroxylapatite gave a preparation with 2.6 $\text{Manduca}$ PTTH units/μg protein (4400-fold purification). Activity was sensitive to proteolytic enzymes. Further purification by preparative electrophoresis gave a preparation which migrated as a single band in two polyacrylamide gel electrophoresis systems. A molecular weight estimate of 25,000 Daltons was obtained for this bands on SDS polyacrylamide gels.     

DNA-binding domains of human fibronectin

Here we report the use of the technique of transferring proteins from polyacrylamide gels to nitrocellulose sheets to identify the DNA-binding domains of human plasma fibronectin. After separation of fibronectin and its tryptic digest on a sodium dodecylsulfate-polyacrylamide gel, the polypeptides were transferred to nitrocellulose sheets and the DNA-binding polypeptides were identified by incubation of the nitrocellul$\text{o}$ se sheets with ³²P-labeled human DNA followed by autoradiography.The following results were obtained: a) only two fibronectin trypsin resistant peptides (50 and 60 kd) showed DNA-binding capacity; b) in competition experiments a 300 fold excess of E. coli DNA did not inhibit the human DNA-human fibronectin binding, indicating the specificity of the interaction.     

Binding of cytochrome c to cytochrome c-oxidase in intact mitochondria. A study with radioactive photoaffinity-labeled cytochrome c

[³H]-p-Azidophenacylbromide-(methyl-4-mercaptobutyrimidate)-cytochrome $\text{c}$ from $\text{Saccharomyces cerevisiae}$ was prepared and its properties determined. The radioactive photoaffinity-labeled cytochrome $\text{c}$ was linked by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase. Analysis of the complex on SDS-polyacrylamide gels showed that cytochrome $\text{c}$ bound to one of the smaller subunits of cytochrome $\text{c}$ oxidase with an apparent molecular weight of 15,000.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Isolation and partial characterization of “galactoprotein a” (LETS) and “galactoprotein b” from hamster embryo fibroblasts

The cell surface glycoproteins of hamster NIL cells, labeled with galactose oxidase and NaB³H4, were selectively solubilized by sequential extraction with Tris buffer containing 1) sucrose-ATP-EDTA, 2) zwitterionic detergent (Empigen BB), and 3) 8 M urea. The previously reported “galactoprotein b” $\text{(Gap b)}$ and “galactoprotein a” ($\text{Gap a}$ or LETS) were isolated by affinity chromatography on insoluble Ricinus communis lectin colums (RCA column) from extracts 2) and 3), respectively. The affinity-purified $\text{Gap a}$ contained an actin-like protein, whereas the other affinity-purified galactoproteins did not contain the actin-like protein. The isolated $\text{Gap b}$ was heterogeneous, and an additional glycoprotein, specific for NILpy cells was copurified on RCA-column with $\text{Gap b}$.